(1) Field of the Invention
This invention relates to a novel plasmid, a method for construction of the plasmid, a novel microorganism containing the plasmid, and a method for cultivation of the microorganism and relates more particularly to a novel plasmid with DNA coding for production and extracellular secretion of such high-molecular substance as xylanase, penicillinase or beta-galactosidase, a novel microorganism transformed with the plasmid, and a method for production and secretion of such high-molecular substance by culturing the microorganism.
(2) Description of the Prior Art
A plasmid is a non-chromosomal gene of cyclic DNA found in a microorganism cell. Plasmids are currently being used as means for recombination of microorganism genes and are becoming more and more important in the fermentation industry.
Studies have recently been done on plasmids carrying foreign DNA having genetic information of metabolic products or specific demand for growth of microorganism, as shown in the production of amino acids or peptides. Some plasmids have been introduced into host microorganisms to obtain transformants. Methods have been proposed for producing relatively low molecular compounds such as amino acids and peptides by culturing the transformants. However, the degree of propagation of plasmids carrying genes for production of high-molecular substances depends on the nature of the host microorganisms and these plasmids have not effectively been expressed. Furthermore, no effective methods for cultivation of such transformants have been established.
It has not been possible to obtain a certain extracellular high-molecular product by culturing a microorganism transformed with a plasmid carrying a foreign DNA fragment having genetic information of extracellular secretion of high-molecular substances which are metabolic products of another microorganism. However, such extracellular secretion has not been successful even using a transformant of Escherichia species, which is the most commonly used host microorganism.
U.S. Pat. No. 4,411,994 of W. Gilbert et al discloses a process for producing specific proteins in bacteria and having them excreted from the bacterial cell. This process comprises inserting the DNA representing the desired non-bacterial protein or part of a protein by recombinant techniques into a plasmid or phage gene for either a periplasmic or an extracellular protein, hereinafter called a "carrier protein", transforming a bacterial host with the recombinant gene, and culturing the transformed host to excrete the protein. The process of this patent provides a means for producing a selected protein by employing a gene for a carrier protein which has a leader sequence of hydrophobic amino acids at its amino terminus.
The cell wall of Escherichia coli, which has often been used for production of useful physiologically active substances, consists of three kinds of membrane: inner membrane, peptide glycan and outer membrane. The space between the inner and outer membrane is called the periplasmic space. The process of U.S. Pat. No. 4,411,994 has succeeded in the excretion of the products within the periplasmic space but not within the culture medium through the outer membrane or outside the bacterial host cell.
The inventors of this invention previously constructed a plasmid carrying a DNA fragment prepared from chromosomal DNA of a Bacillus strain, and coding for production and extracellular secretion of penicillinase. They introduced the plasmid into Escherichia coli HB101 to obtain a transformant, Escherichia coli HB101 (pEAP2), capable of producing and secreting penicillinase (Japanese Patent Unexamined Publication No. 59-162886).
Further, they succeeded in the construction of a plasmid carrying a DNA fragment prepared from chromosomal DNA of a Bacillus strain and coding for production and extracellular secretion of xylanase. They introduced the plasmid into Escherichia coli HB101 to obtain a transformant, Escherichia coli HB101 (pCX311), having an ability to produce and secrete xylanase (Japanese Patent Application No. 58-232508).
By culturing such a strain containing a plasmid carrying a DNA fragment coding for production and secretion of useful physiologically active substances such as enzymes, it is possible to separate and collect the active substances directly from the culture medium in a greater amount and in a more purified form because there is no need for crushing the cells of the strain and there is no saturation of the active substances within the cells.
However, useful physiollogically active substances are ordinarily accumulated mainly within cells and up to now there have been known only restricted numbers of microorganisms which produce and secrete the useful physiologically active substance into media. Therefore, it is very useful from the industrial point of view to establish a method in which it is possible to create a desired strain which produces and secretes a desired useful physiologically active substance into media.